The ability to tightly control solute and water balance during osmotic challenge is an essential prerequisite for cellular life. Osmotic homeostasis is maintained by regulated accumulation and loss of inorganic ions and organic osmolytes. Organic osmolytes play essential roles in protecting renal medullary cells from extreme hypertonic stress and fluctuating extracellular osmolality associated with the urinary concentrating mechanism. While cellular osmoregulation has been studied extensively in a variety of cell types, including kidney cells, major gaps exist in our molecular understanding of this essential process. The nematode C. elegans provides powerful experimental advantages for defining the genetic bases of fundamental biological processes. These advantages include a fully sequenced genome, genetic tractability, and ease and economy of manipulating gene function. Nematodes normally live in soil where environmental variables such as water availability and solute levels change constantly and dramatically. Recently, we demonstrated that C. elegans readily adapts to and survives extreme hypertonic stress. Given its many experimental advantages, C. elegans thus provides an outstanding model system in which to define the genes and genetic pathways responsible for cellular osmoregulation. This R21 grant application addresses stated objectives of the NIDDK PA entitled, "Pilot and feasibility program related to the kidney". We propose to characterize organic osmolyte homeostasis in C. elegans during adaptation to and recovery from hypertonic stress, perform whole genome microarray analyses to identify genes transcriptionally upregulated by hypertonicity, and assess the role of MAPK signaling pathways in cellular osmoregulation using mutant worm strains and RNAi. We will also perform mutagenesis screens to identify genes required for cellular osmoregulation in C. elegans. Powerful forward genetic screening methods have been used with great success to define molecular aspects of osmoregulation in bacteria and yeast, but have not been utilized previously to characterize this process in animal cells. Our proposed investigations represent a novel approach to the molecular study of animal cell osmotic homeostasis. Given the evolutionarily conserved nature of this process, studies in C. elegans will likely provide unique insights into osmoregulation, signaling mechanisms, and cellular stress and damage repair )rocesses in kidney cells as well as other mammalian cell types and tissues.